Thermostable ribonuclease H

ABSTRACT

A substantially pure preparation of thermostable RNase H is disclosed. In one preferable form of the invention, the RNase H is capable of biological activity after incubation at temperatures equal to or greater than 70° C. for at least ten minutes. In another preferable form of the invention, the purification is from Thermus thermophilus or a closely related organism. The present invention is also a method of digesting RNA polymers that are in duplex form with a DNA molecule. The method comprises exposing the duplex to the isolated thermostable RNase H.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology. Morespecifically, the present invention relates to the creation and use ofthe novel enzyme, thermostable ribonuclease H.

BACKGROUND OF THE INVENTION

Ribonuclease H (endoribonuclease H, EC 5.1.26.4, hereafter referred toas RNase H) is an enzyme capable of hydrolyzing an RNA molecule when theRNA molecule is hybridized with a complementary DNA strand. Thebiological role of the enzyme is not known, and hence it is not knownwhether all organisms possess this enzyme.

RNase H is a useful tool in molecular biology research. RNase H is usedfor degrading the RNA strand after first-strand synthesis in theproduction of double-stranded cDNA. Okayama, H., et al. (1982) Mol.Cell. Biol. 2: 161-170.6; Gubler, U., et al. (1983) Gene 25: 263-269.The enzyme can remove poly-(A) tails from messenger RNA if the mRNA isreacted with oligo-dT₁₂₋₁₈. Vournakis, J., et al. (1975) Proc. Natl.Acad. Sci. USA 72: 2959-2963; Davis, R., et al. (1988) Mol. Cell. Biol.8: 4745-4755.

Most importantly, RNase H is used as a diagnostic tool for detectingspecific target DNA sequences in a biological sample by a probeamplification process. Duck, P., et al. (1990) BioTechniques 9: 142-147.In this diagnostic technique, a probe is made of ribonucleoside basesflanked by deoxy-ribonucleoside bases. The probe hybridizes to a targetDNA molecule. RNase H digests the ribonucleoside bases and cleaves theprobe. The probe fragments then dissociate from the target. After manycycles, these fragments accumulate and serve as a basis for detectingthe presence of the target.

RNase H is a component of another diagnostic test called theself-sustained sequence replication (3SR) amplification system, which isa transcription-based amplification method. Guatelli, J. C., et al.(1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878. In the 3SR system, atarget nucleic acid sequence is replicated exponentially by using RNaseH, a DNA-dependent RNA polymerase and reverse transcriptase.

RNase H may also be used to map the location of sequences on an RNAmolecule. First the RNA is annealed with specificoligodeoxyribonucleotide probes and then the duplexed RNA is cleavedwith RNase H. Donis-Keller, H. (1979) Nucleic Acids Res. 7: 179-192.

RNase H may also be used to quantitate poly-(A)-containing mRNA inbiological samples. Krug, M. S., et al. (1987) Methods Enzymol. 152:262-266. RNase H is useful in cDNA cloning via subtractive hybridization(Kzze, K., Shimizu, et al. (1989) Nucleic Acids Res. 17: 807) and forhybrid-arrest translation (Minshull, J., et al (1986) Nucleic Acids Res.14: 6433-6451).

The RNase H enzyme used in the above-mentioned research was isolatedfrom E. coli. The E. coli enzyme is called "RNase HI" and is the productof the rnhA gene. Berkower, I., et al. (1973) J. Biol. Chem. 248:5914-5921; Kanaya, S., et al. (1983) J. Biol. Chem. 258: 1276-1281.RNase H has been identified in other organisms besides E. coli, such asyeast, KB cells, Krebs II ascites cells and avian myeloblastosis virusinfected cells, although its existence in other species is stillunknown. Crouch, R. J. (1981) in Gene Amplification and Analysis(Chirikjian, J. G., and Papas, T. S., eds.) Vol. 2, pp. 218-228,Elseivier, North Holland, N.Y.; Crouch, R. J., et al. (1982) in Nuclease(Linn, S. M., and Roberts, R. J., eds.) pp. 211-241, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. No RNase H has been isolated fromany thermophilic organisms.

A thermostable RNase H would be preferable in many RNase H applications,particularly for mapping and certain diagnostic applications. Inaddition to the obvious advantages of longer reagent shelf life andgreater stability under reaction conditions, a thermostable RNase Hwould allow reactions to be carried out at higher temperatures. Thesehigher temperatures are closer to optimal temperatures for hybridizationof RNA probes to target DNA and would destroy the activity of E. coliRNase H. The optimum temperature for a nucleic acid hybridization willdepend on the hybridization buffer, but typically reaction temperaturesin the 70° C. to 95° C. range facilitate maximum sensitivity by ensuringthat target DNA sequences are melted and accessible and reactiontemperatures in the 45° C. to 75° C. range facilitate maximumselectivity of hybrid formation because there is more hybridizationstringency at higher temperature. High stringency conditions result inlower background by minimizing nonspecific binding of probes tounrelated target sequences.

What is needed in the art of molecular biology is an isolated RNase Hcapable of biological activity at elevated temperatures. Such an RNase Hshould be capable of biological activity after incubation attemperatures greater than 45° C. for at least ten minutes. Preferablythe RNase H will be capable of biological activity after incubation attemperatures of at least 70° C. for at least ten minutes.

SUMMARY OF THE INVENTION

The present invention is a substantially pure preparation ofthermostable RNase H. In one preferable form of the invention, the RNaseH is capable of biological activity after incubation at temperatures ofat least 70° C. for at least ten minutes. In another preferable form ofthe invention, the purification is from Thermus thermophilus HB8 or arelated organism.

The present invention is also a method of digesting RNA polymers thatare in duplex form with a DNA molecule. By "DNA" and "RNA" we meannaturally occurring DNA and RNA and chemically and enzymaticallymodified DNA and RNA. We also mean to include DNA and RNA that issynthetically made and DNA and RNA that contains variant bases. Themethod comprises exposing the duplex to the isolated thermostable RNaseH. In a preferable form of the invention, the DNA molecule is notdigested by the RNase H.

It is an object of the present invention to provide a RNase H capable ofactivity at incubation temperatures close to the optimal temperaturesfor hybridization of RNA probes to target DNA.

It is another object of the present invention to provide an RNase Hcapable of activity after incubation at temperatures greater than 45° C.for at least ten minutes.

It is another object of the present invention to provide an RNase Hcapable of activity after incubation at temperatures of at least 70° C.for at least ten minutes.

It is another object of the present invention to provide a method fordigesting an RNA molecule when the RNA molecule is hybridized to a DNAmolecule.

It is another advantage of the present invention that the RNase H willnot become inactivated when incubated at a temperature of greater than45° C.

It is another advantage of the present invention that the method ofdigesting RNA can take place at higher temperatures.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram of T. thermophilus RNase H on BioRex 70.

FIG. 2 is a chromatogram of T. thermophilus RNase H on Sephacryl 5100HR.

FIG. 3 is a chromatogram of T. thermophilus RNase H on heparin agarose.

FIG. 4 is a chromatogram of T. flavus RNase H on BioRex 70.

DETAILED DESCRIPTION OF THE INVENTION 1. In General

The present invention requires a substantially pure preparation of athermostable RNase H. By "substantially pure" we mean a preparationcapable of digesting an RNA molecule when the RNA molecule is hybridizedto a DNA molecule. The present invention is also a preparation of RNaseH of greater than 0.01 Units/μg (units are defined below). Preferably,the preparation is greater than 1 Units/μg. This preparation should notcontain substantial amounts of other enzymes or constituents that wouldbe detrimental to the RNase H function. By "thermostable" we mean theenzyme will have not lost significant biological activity afterincubation over 45° C. for ten minutes. Preferably the enzyme will havesignificant and useful biological activity after being incubated attemperatures greater than 70° C. for ten minutes. The existence ofthermostable RNase H was previously unknown. Here, we have successfullypurified a thermostable RNase H, thereby proving the existence of suchan enzyme, and also described a methodology for isolation other speciesof RNase H from other thermophilic organisms.

2. Purification of RNase H from a Thermostable Organism

In order to obtain thermostable RNase H from its native host, anappropriate thermostable microorganism must be identified and cultured.Suitable microorganisms are those which contain a thermostable RNase Hcapable of isolation and biological activity. Thermus thermophilus HB8,Thermus aquaticus YT-1, Thermus flavus, and Bacillus stereothermophiluswere discussed in the examples below, but other microorganisms areequally suitable. Organisms that are closely related to Thermusthermophilus HB8, such as Thermus thermophilus HB27, are particularlysuitable. Standard fermentation methods are used to obtain a sufficientamount of the microorganism to isolate the RNase H.

Additionally, the RNase H gene may be cloned from an appropriate source,such as Thermus thermophilus HB8, and expressed in a non-thermostablehost, such as E. coli. Suitable quantities of the enzyme can then beproduced by the heterologous host and the enzyme recovered byconventional means for recovering protein produced in such a host. Anon-thermostable RNase H may be cloned and then selectively mutagenizedto acquire a thermostable enzymatic activity. This could effectively bedone by creating oligonucleotide sequences coding for the portions ofthe thermostable RNase H different from the E. coli enzyme and replacingthose sequences in the E. coli RNase H gene.

The RNase H enzyme can also be isolated from other thermophilicmicroorganisms in an analogous procedure to the one detailed below. Afirst consideration is an assay for the presence of the enzyme indifferent purification fractions. Typically, this is done by examining afraction for the presence of RNase H activity and a protein of theappropriate size.

The RNase digesting ability of RNase H may be detected in many ways. Inthe examples below, we disclose a suitable method of testing for RNase Hactivity. RNase H activity is assayed in a 100 μl reaction mixturecontaining: 1 mM poly-(rA); 100 μM oligo-(dT)₁₈ ; 0.01M Tris-HCl, pH7.5; 10 mM MgCl₂, 0.1 mM EDTA; 1 mM dithiothreitol; 0.1M NaCl; and thepurification fraction. The poly-(rA) and oligo-(dT) molecules will formduplexes. If RNase H is present, the poly-(rA) in the duplex will bedigested and the oligo-(dT) will be free to from another duplex. Thus,the reaction will "cycle". The digested ribonucleosides are acid solubleand may be spectrophotometrically monitored. After incubation at 45° C.for 20 minutes, 1 ml of ice-cold 5% trichloroacetic acid is added to thereaction mixture. After incubation on ice for 5 minutes, the precipitateis removed by microcentrifuge centrifugation. The absorbance of thesupernatant solution is determined at 260 nm in a spectrophotometer. Oneunit of RNase H is defined here as the amount of enzyme generating 1nmole of acid-soluble product in 20 minutes under the above conditions.

Preferably, during the purification all initial steps are conducted at4° C. The chromatographic steps are conducted at room temperature.Purification of RNase H is preferably monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The stainedelectrophoretic gels are monitored for the occurrence of a protein ofthe appropriate size. Thermus thermophilus RNase H is approximately20,000 d. RNases H isolated from other organisms are of comparable size.For instance, E. coli RNase H is approximately 17,500 d.

Preferably, 1 Kg of cells is suspended in a buffer containing 50 mMTris-HCl, pH 7.5; 1.0 mM EDTA; 0.1% (v/v) 2-mercaptoethanol; and 5%(v/v) glycerol (abbreviated as TEBG buffer). The cells are lysed and thelysate is treated with polyethyleneimine to precipitate the nucleicacids. The lysate is centrifuged. The pellet contains nucleic acids andis discarded.

At this point, it is useful to subject the supernatant to ammoniumsulfate precipitation. The protein fraction that precipitates between33% and 45% saturation with ammonium sulfate contains RNase H. Thisprecipitate is collected by centrifugation, dialyzed against TEBG bufferand then dialyzed against TEBG buffer additionally containing 0.05M NaClfor approximately 6 hours or until the sample is the same conductivityas the BioRex 70 chromotography buffer.

After dialysis, the solution is preferably applied to a BioRex 70 columnequilibrated with TEBG/0.05M NaCl buffer. The purpose of the BioRex 70column is to remove contaminating proteins.

The unbound protein is washed from the Bio Rex 70 column with theTEBG/0.05M NaCl buffer. RNase H is eluted from the column with a saltgradient of 0.05-0.5M NaCl. In the example below, a single activity peakwas eluted at approximately 0.3M NaCl, but the elution point might varyfrom preparation to preparation. The fraction containing RNase Hactivity may be located by either an RNase H assay or SDS-PAGE analysis.

Fractions containing RNase H are pooled and the protein is precipitatedwith ammonium sulfate at 66% saturation. This precipitate is collectedby centrifugation and dissolved in a small volume of TEBG buffer.

The dissolved sample is then preferably chromatographed on a SephacrylS-100 HR sizing column. The purpose of this column procedure is toseparate the proteins contained in the sample by size. Fractions aretaken from this column and each fraction analyzed for RNase H activity.Protein concentration in the column fractions may be determined by aprotein assay, such as the Bradford assay. Bradford, M. M. (1976) Anal.Biochem. 72: 248-254. Fractions containing RNase H are pooled anddialyzed against TEBG/0.05 NaCl.

This pooled sample is preferably chromatographed on a heparin agarosecolumn equilibrated with TEBG/0.05 NaCl buffer. The heparin agarosecolumn procedure removes additional protein contaminants. Fractions areassayed for RNase H activity and aliquots are electrophoresed onSDS-PAGE. Fractions containing RNase H are pooled and dialyzed againststorage buffer. The enzyme may then be stored at -20° C. A typicalstorage buffer is 0.05M Tris-HCl, pH 7.5; 0.1 mM EDTA; 1 mMdithiothreitol; 0.1M NaCl, 50% (v/v) glycerol; and 0.1% (v/v) TritonX-100.

3. Thermostability Analysis

A newly isolated RNase H must be analyzed for thermostability to verifythe desired level of that trait. This is most easily done in acomparison test with E. coli RNase H, as described below in theExamples. Basically, the new RNase H and E. coli RNase H areindividually preincubated in a reaction buffer without poly-(rA) andoligo-(dT). RNase H activity is determined after adding poly-(rA) andoligo-(dT). The reaction mixture is incubated at different temperaturesto determine the temperature stability range of the RNase H. In theexamples below, the RNase H digestions were incubated for ten minutes at37° C., 45° C., 70° C. and 90° C. in 50 μl of reaction buffer containing0.01M Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.1 mM EDTA, 1 mM dithiothreitol,and 0.1M NaCl.

After readjusting the incubation temperature back to 20° C., poly-(rA)and oligo-(dT) are added to the enzyme-containing solution and the RNaseH activity determined. An RNase H is "thermostable" if after incubationat temperatures greater than 45° C. for ten minutes, the RNase H iscapable of digesting the added poly-(rA). In the example below, theRNase H from Thermus thermophilus was incubated at 45° C. to obtainoptimum digestion, as compared to 37° C. for E. coli RNase H.

4. Purity of Enzyme

A single unit of RNase H activity was defined above as the amount ofenzyme generating 1 nmole of acid-soluble product in 20 minutes in theassay for RNase H activity described above. By using the purificationprotocol generally described above, it is possible to reliably isolatethermostable RNase H in a purity such that the activity level is above100 units μg total protein. Since as produced in its native organism,the concentration of thermostable RNase H is less than 0.01 units perμg, this represents a concentration of the enzyme in excess of 10,000times over its concentration in the native organism. In general,separated functions recovered from host organisms which have activitylevels in excess of 0.1-1 units per μg total protein would provideuseful levels of enzyme concentrations for at least some molecularbiology procedures. At a purification of 10 units per μg, still ten-foldless than is possible by our purification method, the preparation isuseful for many more molecular biology procedures.

EXAMPLES

1. Purification of RNase H from Thermus thermophilus.

Thermus thermophilus HB8 strain (available without restriction from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.as ATCC No. 27634) was grown in a 300 liter fermenter in the followingmedium: 4 g/L yeast extract; 8 g/L peptone; and 2 g/L NaCl. The pH wascontrolled at 7.5-8.0 with ammonium hydroxide, the dissolved oxygen wasmaintained at 40%, the temperature was maintained at 70° C., and thestirring rate was maintained at 400 rpm. Cells were harvested bycentrifugation at late log phase at a cell density of approximately 5g/L.

RNase H activity was assayed in a 100 μl reaction mixture containing: 1mM poly-(rA); 100 μM oligo(dT)₁₈ ; 0.01M Tris-HCl, pH 7.5; 10 mM MgCl₂,0.1 mM EDTA; 1 mM dithiothreitol; 0.1M NaCl and the purificationfraction. After incubation at 45° C. for 20 minutes, 1 ml of ice-cold 5%trichloroacetic acid was added to the reaction mixture. After incubationon ice for 5 minutes, the precipitate was removed by microcentrifugecentrifugation. The absorbance of the supernatant solution wasdetermined at 260 nm in a spectrophotometer. One unit of RNase H wasdefined as the amount of enzyme generating 1 nmole of acid solubleproduct in 20 minutes under the above conditions.

Purification of RNase H was monitored by tris-glycine, sodium dodecylsulfate polyacrylamide gel electrophoresis (abbreviated as SDS-PAGE).The gels contained 15% acrylamide with an acrylamide/bis-acrylamideratio of 30:1. Gels were electrophoresed, stained in Coomassie brilliantblue, and destained by standard methods, Laemmli, U. K. (1970) Nature277: 680-685. A set of polypeptides (described below) was used as amolecular weight standard.

T. thermophilus RNase H was purified using the following protocol. Allof the purification steps were conducted at 4° C. with the exception ofthe chromatographic steps, which were conducted at room temperature. 1kg of cells was suspended in a buffer consisting of 50 mM Tris-HCl, pH7.5; 1.0 mM EDTA; 0.1% (v/v) 2-mercaptoethanol; and 5% (v/v) glycerol(abbreviated as TEBG buffer). Cells were lysed and the lysate wastreated with polyethyleneimine to precipitate the nucleic acids.Jendrisak, J. (1987) in Protein Purification (Burgess, R. R., ed.), AlanR. Liss, Inc., New York, N.Y., pp. 75-97.

After centrifugation, the protein fraction precipitating between 33% and45% saturation with ammonium sulfate was collected by centrifugation andwas dissolved against TEBG buffer. The resulting solution was dialyzedagainst TEBG buffer containing 0.05M NaCl.

The dialysate was applied to a 2.5-cm×15-cm column of BioRex 70equilibrated with TEBG buffer containing 0.05M NaCl. After washingunbound protein from the column with the same buffer, RNase H was elutedfrom the column with 10 column volumes of a salt gradient of 0.05-0.50MNaCl. Fractions of approximately 20 ml were collected.

A single activity peak was eluted at approximately 0.30M NaCl from thecolumn. The protein was continuously monitored at 280 nm with an IscoModel UA-5 absorbance monitor. FIG. 1 is a chromatogram showing theRNase H peak. Flowthrough and wash fractions are not shown on the FIG. 1chromatogram. 50 μl aliquots were assayed for RNase H activity.Fractions 43-49 were pooled and protein concentrated for subsequentchromatography on Sephacryl S-100 HR. 5 μl aliquots of every fifthcolumn fraction were subjected to an SDS-PAGE analysis along with a laneof polypeptide molecular weight markers. Fractions 45-50 had a proteinband at approximately 20 Kd, suggesting that RNase H was present.

The active fractions (43-49) were pooled and the protein wasprecipitated with ammonium sulfate at 66% saturation. The precipitatewas collected by centrifugation and was dissolved in a small volume ofTEBG buffer. The protein sample was then chromatographed on a2.5-cm×122-cm Sephacryl S-100 HR sizing column equilibrated withTEBG/0.5M NaCl. Fractions of approximately 20 ml were collected. Asingle peak of RNase H activity eluted at approximately 1.6 times thevoid volume of the column.

FIG. 2 is a chromatogram showing the RNase H peak. The proteinconcentration in the column fractions was determined by the Bradforddye-binding assay, Bradford, M. M. (1976) Anal. Biochem. 72: 248-254,and the RNase H activity was determined with 10 μl aliquots of columnfractions. RNase H activity was detected in fractions 29 and 30. Thesefractions were pooled for dialysis. An SDS-PAGE analysis was performedon 5 μl aliquots of fractions. Fraction 29 and 30 showed a band at 20Kd.

The pooled fractions were dialyzed against TEBG buffer containing 0.05MNaCl. The sample was chromatographed on a 1.5-cm×10-cm heparin agarosecolumn equilibrated with TEBG buffer containing 0.05M NaCl. FIG. 3 is achromatogram that shows the results of this procedure. RNase H waseluted from the column with the same buffer and contaminants were elutedwith 10 column volumes of a salt gradient of 0.05-0.25M NaCl. Fractionsof approximately 4 ml were collected.

Still referring to FIG. 3, protein was continuously monitored at 280 nmwith an Isco Model UA-5 absorbance monitor and RNase H activity wasdetermined with 10 μl aliquots of column fractions as described above.Additionally, an SDS-PAGE analysis was performed on 5 μl aliquots of theheparin agarose column fractions. Fractions 8-16 contained a singlepolypeptide band of molecular weight 20,000 d. These fractions exactlycorrelated with RNase H activity in the column fractions. Fractions 7-16were pooled and dialyzed against storage buffer prior to storage ofenzyme at -20° C. The fractions were dialyzed for 16 hours at 4° C.against a storage buffer containing 0.05M Tris-HCl, pH 7.5; 0.1 mM EDTA;1 mM dithiothreitol; 0.1M NaCl, 50% (v/v) glycerol; and 0.1% (v/v)Triton X-100.

2. Characterization of the Purified Enzyme

The molecular weight of T. thermophilus RNase H was determined bySDS-PAGE. One μg of the protein and each marker was subjected toanalysis. Marker proteins were phosphorylase b (94,000), bovine serumalbumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000),soybean trypsin inhibitor (20,100), and lactalbumin (14,400). The datafrom the SDS-PAGE indicated that T. thermophilus RNase H has a molecularweight of about 20,000. This is similar to the molecular weight of E.coli RNase HI. E. coli RNase H1 has a published molecular weight of17,559 based on the derived amino acid sequence. Kanaya, S. et al.(1983) J. Biol. Chem. 258: 1276-1281.

Amino acid analysis of the isolated RNase was performed on an AppliedBiosystems 420-A-03 micro amino acid analyzer/derivatizer. Cysteineresidues were first pyridylethylated after the protein was fullydenatured and any disulfides reduced. The amino acid composition ofThermus thermophilus RNase H is presented and compared to thecomposition of E. coli RNase H in Table 1. The composition of the E.coli enzyme was derived from the DNA sequence of the rnh A gene. Kanaya,S. et al. (1983) J. Biol. Chem. 258: 1276-1281. Amino acid compositionindicates that the molecular weight of Thermus thermophilus RNase H is20,018, which is in good agreement with the value derived from SDS-PAGE.

N-terminal microsequencing was done on the Applied Biosystems model 475vapor phase protein sequence. PTH-amino acids were analyzed with anon-line microbore HPLC. Data were recovered and reduced with an AppliedBiosystems 900-A data workstation and sequence-dedicated software. Thesequence is compared to that of E. coli RNase HI in Table 2 andindicates some homology, especially in a 14 amino acid block starting atresidue number 12 in the Thermus thermophilus RNase H enzyme. Table 2 isa comparison of these segments. In Table 2, exact matches are indicatedby boldface, and conservative differences are underlined. The sequencedata for E. coli RNase HI is from Kanaya and Crouch (1983) J. Biol.Chem. 258: 1276-1281.

                  TABLE 1                                                         ______________________________________                                        Amino acid composition of Thermus thermohilus RNase H                                      Comp.   # Residues                                               Amino Acid                                                                              Abbrev.  by MW     T. thermophilus                                                                         E. coli                                ______________________________________                                        Aspartic acid +                                                                         Asx      13.863    14        14                                     Asparagine                                                                    Glutamic acid +                                                                         Glx      21.715    22        20                                     Glutamine                                                                     Serine    Ser      4.820     5         4                                      Glycine   Gly      17.635    18        14                                     Histidine His      6.273     6         5                                      Arginine  Arg      16.187    16        10                                     Threonine Thr      7.887     8         10                                     Alanine   Ala      22.308    22        14                                     Proline   Pro      14.100    14        5                                      Tyrosine  Tyr      2.500     3         5                                      Valine    Val      7.382     7         9                                      Methionine                                                                              Met      3.254     3         4                                      Cysteine  Cys      3.622     4         3                                      Isoleucine                                                                              Ile      1.867     2         7                                      Leucine   Leu      19.596    20        12                                     Phenylalaine                                                                            Phe      6.965     7         2                                      Lysine    Lys      9.807     10        11                                     ______________________________________                                         Calculated MW of sample: 20,018                                          

                                      TABLE 2                                     __________________________________________________________________________    N-terminal amino acid sequence of Thermus thermophilus RNase                  __________________________________________________________________________    H.                                                                             1  2  3  4  5  6  7  8  9 10 11 12 13 14 15                                  met met                                                                          asn                                                                              pro                                                                              ser                                                                              pro                                                                              arg leu                                                                          lys lys                                                                          arg gln                                                                          val val                                                                          ala glu                                                                           ##STR1##                                                                        phe phe                                                                          thr thr                                                                          asp asp                                                                          gly gly                             16 17 18 19 20 21 22 23 24 25 26 27 28 29 30                                   ##STR2##                                                                        cys cys                                                                          leu leu                                                                          gly gly                                                                          asn asn                                                                          pro pro                                                                          gly gly                                                                          pro pro                                                                          gly gly                                                                          gly gly                                                                          cys tyr                                                                           ##STR3##                                                                        arg  ala                                                                          ##STR4##                                                                        leu leu                             31 32 33 34                                                                   arg arg                                                                           ##STR5##                                                                         ##STR6##                                                                         ##STR7##                                                                              Thermus thermophilus HB-8 E. coli                           __________________________________________________________________________

3. Heat stability comparison of Thermus thermophilus and E. coli RNasesH

T. thermophilus and E. coli RNases H were preincubated in a reactionbuffer minus poly-(rA) and oligo-(dT). The residual nuclease activitieswere determined after adding poly-(rA) and oligo-(dT).

10 units of Thermus thermophilus and E. coli RNases H were incubated for10 minutes at 37° C., 45° C., 70° C., and 90° C. in 50 microliters ofreaction buffer containing 0.01M Tris-HCl, pH 7.5, 10 mM MgCl₂ ; 0.1 mMEDTA, 1 mM dithiothreitol, and 0.1M NaCl. After readjusting theincubation temperatures back to 20° C. by incubation for 10 minutes in a20° C. water bath, 50 microliters of 2 mM poly-(rA) and 0.2 mMoligo-(dT₁₈) in reaction buffer were added to the enzyme. Afterincubation for 45° C. for 20 minutes in the case of T. thermophilusRNase H and 37° C. for 20 minutes in the case of E. coli RNase H, thereactions were stopped and undigested poly-(rA) was precipitated bytrichloroacetic acid precipitation. After centrifugation to pellet theundegraded poly-(rA), absorbance at 260 nm was determined for thesupernatant solutions in order to determine the amount of poly-(rA) thathad been digested. The absorbance values were corrected for backgroundabsorbance in a minus enzyme control and the values were normalized tothe activity of E. coli RNase H and Thermus thermophilus RNase H whichwere not subjected to the heat treatment protocol. These values were setat 100% activity and the heat-treated activities are expressed as apercentage of these control values. The results (Table 3) indicate thatThermus thermophilus RNase H is stable upon heating, even up to 90° C.for 10 minutes, whereas the RNase HI from E. coli begins to loseactivity at 45° C. for 10 minutes.

                  TABLE 3                                                         ______________________________________                                        Thermostability comparison of RNases H from                                   E. coli and Thermus thermophilus                                                             Residual Activity (%)                                          Preincubation conditions                                                                       E. coli T. thermophilus                                      ______________________________________                                        10 min., 37° C.                                                                         100     100                                                  10 min., 45° C.                                                                         98      101                                                  10 min., 70° C.                                                                         8        98                                                  10 min., 90° C.                                                                         O        97                                                  ______________________________________                                    

4. Screening for thermostable RNase H from other thermophilic organisms

A protein extract was prepared from the thermophilic organism Thermusflavus and was subjected to BioRex 70 column chromatography as describedabove. Fractions were assayed for RNase H activity. The elution profileis shown in FIG. 4. As with Thermus thermophilus, a single peak of RNaseH activity was detected eluting at about 0.30M NaCl from the BioRex 70column. The peak fraction was reassayed after preincubating aliquots ofthe peak fraction for 10 minutes at (A) 45° C., (B) 70° C. and (C) 90°C., as described in Table 3, and the residual RNase H activity wasdetermined. At all pretreatment temperature conditions, the RNase Hactivity from T. flavus was stable and thus resembled the thermostableRNase H activity isolated from Thermus thermophilus HB8. The pointlabelled "D" in the graph is the RNase H activity when the assay wasdone in the absence of 100 μM oligo-(dT). The lack of poly-(A) digestionunder these conditions confirms that this activity is indeed RNase H.

These experiments demonstrate that an analogous thermostable RNases Hcan be isolated from other thermophilic organisms. Organisms such asThermus aquaticus, Bacillus stereothermophilus and bacteria isolatedfrom deep-sea thermal vents (e.g. Thermococcus litoralis), using anapproach similar to the isolation of RNase H from Thermus thermophilusmay also be suitable for the isolation of thermostable RNase H.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Thermus thermophilus                                           (B) STRAIN: HB-8                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetAsnProSerProArgLysArgValAlaLeuPheThrAspGlyAla                              151015                                                                        CysLeuGlyAsn ProGlyProGlyGlyCysAlaArgLeuLeuArgPhe                             202530                                                                        LysAla                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acids                                                          (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (v) FRAGMENT TYPE: N-terminal                                                 (vi) ORGINAL SOURCE:                                                          (A) ORGANISM: Escherichia coli                                                (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Ranaya, S.                                                       (C) JOURNAL: J. Biol. Chem.                                                   (D) VOLUME: 258                                                               (F) PAGES: 1276-1281                                                          (G) DATE: 1983                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       Met LeuLysGlnValGluIlePheThrAspGlySerCysLeuGlyAsn                             151015                                                                        ProGlyProGlyGlyTyrGlyAlaIleLeuArgTyrArgGly                                     2025                                                                     

We claim:
 1. An essentially pure thermostable RNase H wherein the RNaseH has a molecular weight of approximately 20,000 daltons and is capableof digesting added poly(rA) after incubation at 70° C. for 10 minutesand wherein the digestive capability after incubation at 70° C. for 10minutes is at least about 98% of the digestive capability afterincubation at 37° C. for 10 minutes.
 2. The RNase H of claim 1 whereinthe RNase H is isolated from Thermus thermophilus.
 3. A process ofdigesting RNA in an RNA-containing molecule, wherein the RNA molecule isin duplex form with a DNA molecule, comprising exposing the nucleic acidduplex under conditions suitable for enzyme activity to an effectiveamount of an essentially pure preparation of a thermostable RNase suchthat the RNA is digested, wherein the RNase has a molecular weight ofapproximately 20,000 daltons and is capable of digesting added poly-(rA)after incubation at 70° C. for 10 minutes and wherein the digestivecapability after incubation at 70° C. for 10 minutes is at least about98% of the digestive capability after incubation at 37° C. for 10minutes.